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United States Patent | 5,606,023 |
Chen ,   et al. | February 25, 1997 |
Mutant tumor necrosis factor proteins which retain full or near full capability to bind to a TNFR-p75 receptor while retaining only a limited capability to bind a TNFR-p55 receptor are provided. Methods of inhibiting toxicity induced by tumor necrosis factor by treating cells or tissues having a tumor necrosis factor receptor with these mutant human tumor necrosis factor proteins are also provided. Pharmaceutical compositions and methods of inhibiting systemic tumor necrosis factor induced toxicity in a patient undergoing antitumor TNF-.alpha. therapy with these compositions are also provided.
Inventors: | Chen; Mann-Jy (Wayne, PA); Weber; Irene T. (Haverford, PA) |
Assignee: | Thomas Jefferson University (Philadelphia, PA) |
Appl. No.: | 249190 |
Filed: | May 24, 1994 |
Current U.S. Class: | 530/351; 424/85.2; 435/69.52 |
Intern'l Class: | C07K 014/525; A61K 038/19; C12N 015/28 |
Field of Search: | 435/69.52 424/85.1,85.2 514/2,8,12 530/350,351 |
Foreign Patent Documents | |||
0810224 | Mar., 1993 | CA. |
Gennaro, Alfonso, Ed., Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co., Easton, PA, 1990. Goodwin et al., "Molecular cloning and expression of the type 1 and type 2 murine receptors for tumor necrosis factor", Mol. Cell Biol. 1991, 11:3020-3026. Gray et al., "Cloning of human tumor necrosis factor (TNF) receptor cDNA and expression of recombinant soluble TNF-binding protein", Proc. Natl. Acad. Sci. USA 1990, 87:7380-7384. Kunkel T. A., "Rapid and efficient site-specific mutagenesis without phenotypic selection", Proc. Nat'l. Sci. USA 1985 82:488-492. Kunkel T. A. et al., "Rapid and efficient site-specific mutagenesis without phenotypic selection", Methods in Enzymol., 154:367-382. Loetscher et al., "Molecular cloning and expression of the human 55 kd tumor necrosis factor receptor", Cell 1990, 61:351-359. Loetscher et al., "Human Tumor Necrosis Factor .alpha.(TNF.alpha.) Mutants with Exclusive Specificity for the 55-kDA or 75-kDA TNF Receptors", J. Biol. Chem., 1993, 268(35):26350-26358. Schall et al., "Molecular cloning and expression of a receptor for tumor necrosis factor", Cell 1990, 61:361-370. Shortle D. et al., "Local mutagenesis: A method for generating viral mutants with base substitutions in preselected regions of the viral genome", Proc. Nat'l, Sci. USA 1978, 75:2170-2174. Smith et al., "A receptor for tumor necrosis factor defines an unusual family of cellular and viral proteins", Science 1990, 248:1019-1023. Van Ostade et al., "Human TNF mutants with selective activity on the p55 receptor", Nature 1993, 361:266-268. Zhang et al., "Site-directed Mutational Analysis of Human Tumor Necrosis Factor-.alpha. Receptor Binding Site and Structure-Functional Relationship", J. Biol. Chem. 1992, 267(33):24069-24075. Schulz & Schirmer, published 1979, by Springer-Verlag New York, Inc., pp. 14-16. Barbara et al. (1994), The EMBO Journal, vol. 13, pp. 843-850. Varr Ostade et al. (1994) Eur. J. Biochem, vol. 220, pp. 771-779. Bouie et al. (1990), Science vol. 247 pp. 1306-1310. |
__________________________________________________________________________ 5' CTAAGGAAAT ACTTACATAT GGTACGTTCT TCTTCTCGTA CTCCGAGTGA CAAGCCTGT 3' (primer 1; SEQ ID NO: 1) 5' TTAAAGTTCT AAGCTTGGGT 3' (primer 2; SEQ ID NO: 2) __________________________________________________________________________
TABLE 1 ______________________________________ Summary of in vitro binding experiments Ratio TNFR-p55 TNFR-p75 (TNFR-p75/ MUTANT Binding Binding TNFR-p55) ______________________________________ Wild type 100% 100% 1 A33-Asn 10% 109% 11 A33-Gln 13% 14% 1.1 A33-Ile 3% 101% 34 A33-Val 0.6% 44% 73 A33-Met 67% 119% 1.8 A33-Lel 17% 105% 6 ______________________________________
__________________________________________________________________________ SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 3 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 59 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: CTAAGGAAATACTTACATATGGTACGTTCTTCTTCTCGTACTCCGAGTGA50 CAAGCCTGT59 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: TTAAAGTTCTAAGCTTGGGT20 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 61 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: TCGACATGCATTTATTTGCATACATTCAATCAATTGTTATCTAAGGAAAT50 ACTTACATATG61 __________________________________________________________________________